Real time PCR is routinely used for detection of nucleic acids of interest in a biological sample. For a review of real time PCR see, e.g., M Tevfik Dorak (Editor) (2006) Real-time PCR (Advanced Methods) Taylor & Francis, 1st edition ISBN-10: 041537734X ISBN-13: 978-0415377348, and Logan et al. (eds.) (2009) Real-Time PCR: Current Technology and Applications, Caister Academic Press, 1st edition ISBN-10: 1904455395, ISBN-13: 978-1904455394. For additional details, see also, e.g., Gelfand et al. “Homogeneous Assay System Using The Nuclease Activity of A Nucleic Acid Polymerase” U.S. Pat. No. 5,210,015; Leone et al. (1995) “Molecular beacon probes combined with amplification by NASBA enable homogenous real-time detection of RNA.” Nucleic Acids Res. 26:2150-2155; and Tyagi and Kramer (1996) “Molecular beacons: probes that fluoresce upon hybridization” Nature Biotechnology 14:303-308. Traditionally, single well multiplexing, used to detect more than one target nucleic acid per sample in a single reaction container (e.g., well of a multiwell plate), is achieved using self-quenched PCR probes such as TAQMAN™ or Molecular Beacon probes that are specific for each amplicon. Upon binding to the amplicon in solution, or upon degradation of the probes during PCR, the probes unquench, producing a detectable signal. The probes are labeled with fluorophores of different wavelengths, permitting a multiplexing capability of up to about 5 targets in a single “one pot” reaction. More than about 5 probes per reaction is difficult to achieve, due to practical spectral range and label emission limitations. This severely limits multiplexing of a single reaction, which, in turn, significantly limits how many targets can be screened per sample and drives up reagent cost and instrument complexity in detecting multiple targets of interest.
Nucleic acid arrays represent another approach to multiplexing the detection of amplification products. Most typically, amplification reactions are performed on a sample, and amplicons are separately detected on a nucleic acid array. For example, Sorge “Methods for Detection of a Target Nucleic Acid Using A Probe Comprising Secondary Structure” U.S. Pat. No. 6,350,580 propose the capture of a probe that is released upon amplification by purifying the probe out of the amplification mixture and then detecting it. This multiple-step approach to making and detecting amplicons makes real time analysis of the amplification mixture impractical.
Various approaches that amplify the reactants in the presence of the capture nucleic acids have also been proposed. For example, Kleiber et al. “Integrated Method and System for Amplifying And Detecting Nucleic Acids,” U.S. Pat. No. 6,270,965, propose detection of an amplicon via evanescence induced fluorescence. Similarly, Alexandre et al “Identification and Quantification of a Plurality of Biological (Micro) Organisms or Their Components,” U.S. Pat. No. 7,829,313, proposes detection of amplicons on arrays. In another example, target polynucleotides are detected by detecting a probe fragment that is produced as a result of amplification, e.g., by binding to an electrode, followed by electrochemical detection. See, e.g., Aivazachvilli et al. “Detection of Nucleic Acid Amplification” US 2007/0099211; Aivazachvilli et al. “Systems and Methods for Detecting Nucleic Acids” US 2008/0193940, and Scaboo et al. “Methods And Systems for Detecting Nucleic Acids” US 2008/0241838.
These methods all suffer from practical limitations that limit their use for multiplex target nucleic acid detection. For example, Kleiber (U.S. Pat. No. 6,270,965) relies on evanescence induced fluorescence to detect fluorescence of amplicons at the array surface, and requires complex and expensive optics and arrays. Alexandre (U.S. Pat. No. 7,829,313) propose detection of amplicons on an array; as in Kleiber this increases array costs significantly, because each array has to be custom designed to detect each amplicon. In practice, it can be difficult to achieve similar hybridization kinetics for disparate amplicons on an array, particularly where the amplicons are relatively large, as in Alexandre. Furthermore, this art provides little guidance regarding how to detect signal on an array where there is an accompanying solution phase that also comprises high levels of signal background, or of arrays that remain stable through in situ thermal cycling.
The present invention overcomes these and other problems in the art. A more complete understanding of the invention will be obtained upon complete review of the following.